The Human Immunodeficiency Virus (HIV) has long been thought to enter target cells by fusing with the plasma membrane. This notion is based, in part, on the fact that the HIV Env glycoprotein engages CD4 and coreceptors, CCR5 or CXCR4, on the cell surface and then promotes membrane fusion by undergoing pH- independent conformational changes. Our recent study has challenged this view by presenting direct evidence for HIV entry via an endocytic pathway. The arguments for this entry route are based on: (i) the delayed release of HIV content into the cytosol relative to the acquisition of resistane to a membrane-impermeant fusion inhibitor; and (ii) single HIV imaging which reveals complete fusion with endosomes but only partial fusion at the cell surface. However, the notion of HIV entry via endocytosis has not been widely accepted in the field. We therefore propose to carefully evaluate the HIV entry routes in different cell types and define the viral and cellular determinants of the sites of HIV fusion. Our central hypothesis is that HIV can promote only the early steps of fusion, while relying on the host cell to complete this process. This hypothesis stems from the idea that a handful of Env on HIV particles may not be sufficient to overcome a large energy barrier associated with creating the highly unfavorable lipid intermediates en route to fusion. The proposed model makes testable predictions that will guide our quest for the host factors that can aid the HIV fusion. We will: 1. Examine the Env- and cell type-dependence of HIV entry routes. The HIV fusion sites in lymphoid cell lines and primary CD4+ T cells will be defined using an improved single virus imaging approach. 2. Determine whether complete HIV fusion with the plasma membrane requires an external force. We will evaluate the dependence of HIV-cell fusion and of HIV-mediated cell-cell fusion on actin remodeling which can generate lateral membrane tension and thereby promote the dilation of a fusion pore. 3. Explore cellular factors responsible for HIV fusion with endosomes. We will follow up on our pilot data implicating several host proteins in HIV trafficking and fusion. A common feature of the selected host factors is that they generate a membrane curvature or modify the lipid composition and can thus favor HIV-endosome fusion. The proposed studies will define the HIV entry and fusion pathways. We expect to elucidate the extent of virus' reliance on host factors and delineate the mechanism of complete HIV fusion that culminates in the release of the nucleocapsid. PUBLIC HEALTH RELEVANCE: The Human immunodeficiency virus (HIV) deposits its genome into a host cell by fusing its envelope with the cell membrane. Whether the HIV directly fuses at the cell surface upon engaging the requisite receptor and coreceptor or enters the cell and fuses with intracellular compartments is a subject of debate. To define the sites of HIV entry and to identify the cellular targets for therapeutic intervention, we will employ an array of innovative approaches, including single virus imaging and the targeted inactivation of host factors that may be involved in HIV fusion.